Method for measuring ion channel activity

ABSTRACT

The present invention discloses a human embryo kidney cell or cell line transfected with a GABA-gated chloride channel derived from an insect of the order of Lepidoptera, Diptera, Coleoptera, Homoptera, Acarina, Thysanaptera, Heteroptera, Hymenoptera or Isoptera and its use in a one-pot, high-throughput method for the measurement of the change in membrane potential in a cell or cell line. The method is particularly effective in measuring changes in membrane potential resulting from a compound acting on a GABA-gated chloride channel derived from a tobacco budworm.

This is a nonprovisional of provisional application Ser. No. 60/377,089,filed on May 1, 2002.

FIELD OF THE INVENTION

The present invention relates generally to the field of ion channelactivity. In particular, the invention relates to measuring thepotential pesticidal activity of a compound by measuring membranepotential levels in cells or cell lines following chemical treatment,and more particularly it pertains to measuring the membrane potential incells or cell lines transfected with a GABA-gated chloride channelfollowing chemical treatment.

BACKGROUND OF THE INVENTION

Gamma amino-n-butyric acid (“GABA”) gated chloride channels, which arereceptors for GABA, play important roles in inhibiting synaptictransmission in both vertebrate and invertebrate nervous systems. Manyexisting biologically active compounds, for example insecticides, targetthis ion channel. Many of these compounds were initially identifiedbased on their ability to act on the GABA receptor. Compounds directedto this site have quick modes of action. As such, there is a desire todevelop ways to target this channel as a means for identifyingbiologically active compounds, including insecticides.

Cells or cell lines expressing insect ion channel receptors, inparticular insect GABA-gated chloride channels, have been reported inthe literature, for example Tomalski a et al., U.S. Pat. No. 5,854,002;Lee et al., FEBS Lett., 335(3), pp. 315-318 (1993); Shotkoski et al.,FEBS Lett., 380(3), pp. 257-262 (1996); Buckingham et al.,Neuropharmacology, 35(9/10), pp. 1393-1401 (1996); Smith et al., J.Recept. Signal Transduction Res., 15(1-4), pp. 33-41 (1995); Millar etal., Proc. R. Soc. London, Ser B. 258(1353) pp. 307-14 (1994); Hallinget a., U.S. Pat. No. 6,329,516 B1; and Shotkoski et al., Insect Mol.Biol. 3(4) pp. 283-7 (1994). However, most of these cell lines aredifficult to grow, are less stable, may not be conducive for use inhigh-throughput methods for measuring membrane potential. In addition,most of these cell lines tend to involve insect cell lines expressingthe insect GABA receptor which is usually derived from the order ofAedes, Spdoptera, Trichoplusia, or Drosophila, resistant to dieldrine,or is a ligand-gated chloride channel homologue 3, rather than humanembryo kidney (“HEK”) cell lines which express an insect GABA receptorderived from the order of Lepidoptera, Diptera, Coleoptera, Homoptera,Acarina, Thysanaptera, Heteroptera, Hymenoptera or Isoptera. As aresult, there is a need for cells or cell lines that are easy to grow,are stable, conducive for use in high-throughput methods for measuringchanges in membrane potential, and involve human embryo kidney celllines rather than insect cell lines that express the insect GABAreceptor.

Methods for measuring the ability of a compound to act on an insect GABAreceptor are known to one skilled in the art. For example, one ofordinary skill in the art would know that electrophysiology measurementsin oocytes expressing a functional channel can be used to test forinsecticidal activity. Also, see Kellerman et al., WO 01/49848, Hallinget al., U.S. Pat. No. 6,329,516 B1, and Tomalski et al., U.S. Pat. No.5,854,002. However, most of these methods tend to involve cell linesthat are difficult to grow, are less stable, and incorporate insect celllines expressing the insect GABA receptor which is usually derived fromthe order of Aedes, Spdoptera, Trichoplusia, or Drosophila, resistant todieldrine, or is a ligand-gated chloride channel homologue 3. Thesemethods also tend not to be conducive for use in high-throughput methodsgiven that they tend to incorporate a radioisotope or a ligandradiolabeled with a detectable isotope, which require special handlingand can be expensive; measure membrane potential by electrophysiologicalmethods rather than through fluorescence; use multiple reaction vessels;and be time-intensive limiting the number of measurements that can becarried out in a given time period. As such, these methods preclude ahigh-throughput method for measuring changes in membrane potential in aHEK cell or cell line transfected with a GABA-gated chloride channelderived from insects of the order of Lepidoptera, Diptera, Coleoptera,Homoptera, Acarina, Thysanaptera, Heteroptera, Hymenoptera or Isopterathat uses fluorescence. In addition, high-throughput methods reported inthe literature, for example Molecular Devices Corporation's commerciallyavailable FLIPR® Membrane Potential Assay Kit (available from MolecularDevices, Sunnyvale Calif.) are not readily adaptable to allcircumstances; tend to be less sensitive, not as reproducible. As aresult, there is a need for an inexpensive, high-throughput method formeasuring changes in membrane potential via fluorescence in a HEK cellor cell line expressing a GABA receptor gene.

SUMMARY OF THE INVENTION

One embodiment of the present invention describes a cell or cell lineuseful in measuring a membrane potential change in a testing medium. Thepresent invention comprises a HEK cell or cell line containing aGABA-gated chloride channel derived from an insect, preferably insectsof the order of Lepidoptera, Diptera, Coleoptera, Homoptera, Acarina,Thysanaptera, Heteroptera, Hymenoptera or Isoptera.

Another embodiment of the present invention describes a cell or cellline comprising a HEK cell or cell line transfected with a tobaccobudworm GABA-gated chloride channel, wherein the tobacco budwormGABA-gated chloride channel has the nucleotide sequence described inSEQ. ID NO: 4 or the amino acid sequence described in SEQ. ID NO: 5.

Yet another embodiment of the present invention describes ahigh-throughput method for measuring membrane potential. The presentinvention measures changes in fluorescence in response to the additionof a test compound. The present invention is particularly effective inmeasuring changes in fluorescence in the cells or cell lines describedabove.

In yet another embodiment of the present invention, a high-throughputmethod of identifying compounds that decrease fluorescence by comparingtest compounds to a test medium alone or to the test medium followingchemical treatment with compounds that decrease fluorescence isdisclosed. This method can be useful in identify compounds suspected ofexhibiting insecticidal activity.

In still yet another embodiment of the present invention, ahigh-throughput method of identifying compounds with insecticidalactivity through the decrease in fluorescence in the cells or cell linesdescribed above is disclosed.

The present invention is a high-throughput method that is less complex,more cost effective, and comparable in sensitivity to those disclosed inthe art.

DEFINITIONS

The term “ambient temperature” as utilized herein shall mean anysuitable temperature found in a laboratory or other working quarter, andis generally not below about 15° C. nor above about 30° C.

The term “testing vessel” as utilized herein shall mean any device, suchas a petri-dish, a microtiter plate, a test-tube, or beaker, which maybe utilized to perform an assay, a reaction, a method, an experiment, orother procedure.

As used herein, the term “membrane potential indicator” shall mean anysubstance, such as a fluorescent dye, that is capable of indicatingmembrane potential change via fluorescence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the level of fluorescence of a human embryo kidney cellline transfected with a nucleotide sequence (SEQ. ID NO: 4) of a tobaccobudworm following the standard protocol for the FLIPR® MembranePotential Assay Kit.

FIG. 2 shows the level of fluorescence of a human embryo kidney cellline transfected with a nucleotide sequence (SEQ. ID NO: 4) of a tobaccobudworm utilizing the present invention.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention involves a cell or cell linecomprising a human embryo kidney (“HEK”) cell or cell line transfectedwith a GABA-gated chloride channel derived from an insect. Preferably,the insect is of the order of Lepidoptera, Diptera, Coleoptera,Homoptera, Acarina, Thysanaptera, Heteroptera, Hymenoptera or Isoptera,more preferably of the order Lepidoptera or Homoptera, even morepreferably of the order of Lepidoptera. Examples of insects that can beused in the present invention are European corn borers, cutworms,armyworms, tobacco budworms, seed maggots, mosquitoes, nuisance flies,beetles, rootworms, leafhoppers, aphids, mites, whiteflies, scales,stink bugs, thrips, wasps, ants, fruit worms, cabbage worms, moths,loppers, Lygus bugs, weevils, midges, leafminers, leafbeetles, ortermites. Preferred insects are tobacco budworms and aphids.Particularly preferred insects are tobacco budworms. Examples of TBWGABA-gated chloride channels that can be used in the present inventionare the nucleotide sequences described in SEQ. ID NO: 1, SEQ. ID NO: 4,or SEQ. ID NO: 7, preferably SEQ ID NO: 4, or the amino acid sequencesdescribed in SEQ. ID NO: 2, SEQ. ID NO: 5, or SEQ. ID NO: 8, preferablySEQ. ID NO: 5, as set forth herein and disclosed in U.S. Pat. No.6,329,516 B1.

Another embodiment of the present invention describes a HEK cell or cellline transfected with a TBW GABA-gated chloride channel, wherein thetobacco budworm GABA-gated chloride channel has the nucleotide sequenceof SEQ. ID NO: 4 or the amino acid sequence of SEQ. ID NO: 5, as setforth herein and disclosed in U.S. Pat. No. 6,329,516 B1.

Yet another embodiment of the present invention involves a method formeasuring changes of membrane potential in the cell(s) or cell line(s)described above following chemical treatment, the method comprising:

-   -   (a) contacting a fixed amount of a membrane potential indicator        with the test medium in a testing vessel;    -   (b) maintaining the membrane potential indicator in contact with        the test medium in the testing vessel for a time sufficient to        allow the membrane potential indicator to interact with the test        medium;    -   (c) adding a test compound to the testing vessel;    -   (d) adding a fixed amount of GABA, wherein both the GABA and        test compound desire to act on the GABA-gated chloride channel        in the test medium; and    -   (e) measuring the level of fluorescence of the test medium,        wherein the level of fluorescence is inversely proportional to        the amount of the test compound acting on the GABA-gated        chloride channel in said testing medium.

As set forth above, insects of the order of Lepidoptera, Diptera,Coleoptera, Homoptera, Acarina, Thysanaptera, Heteroptera, Hymenopteraor Isoptera, preferably, of the order of Lepidoptera or Homoptera, morepreferably Lepidoptera, may be used in this embodiment. As set forthabove, examples of insects that are useful include, but are not limitedto, European corn borers, cutworms, armyworms, tobacco budworms, seedmaggots, mosquitoes, nuisance flies, beetles, rootworms, leafhoppers,aphids, mites, whiteflies, scales, stink bugs, thrips, wasps, ants,fruit worms, cabbage worms, moths, loppers, Lygus bugs, weevils, midges,leafminers, leafbeetles, or termites. Preferred insects are tobaccobudworms and aphids. Particularly preferred insects are tobaccobudworms. As set forth above, the GABA-gated chloride channel may havethe nucleotide sequences of SEQ. ID NO: 1, SEQ. ID NO: 4, or SEQ. ID NO:7, preferably SEQ. ID NO: 4, or the amino acid sequences of SEQ. ID NO:2, SEQ. ID NO: 5 or SEQ. ID NO: 8, preferably SEQ. ID NO: 5.

In preferred embodiments of the present invention, the GABA-gatedchloride channel is a TBW GABA-gated chloride channel, wherein the TBWGABA-gated chloride channel has the nucleotide sequence of SEQ. ID NO: 4or the amino acid sequence of SEQ. ID NO: 5.

Preferably, the test medium is a cell line transfected with a GABA-gatedchloride channel derived from an insect. More preferably, the testmedium is an HEK cell line transfected with a TBW GABA-gated chloridechannel. Even more preferably, the test medium is an HEK cell linetransfected with a TBW GABA-gated chloride channel wherein the TBWGABA-gated chloride channel has the nucleotide sequence of SEQ. ID NO: 4or the amino acid sequence of SEQ. ID NO: 5.

Examples of membrane potential indicators that may be used in thepresent invention include, but are not limited to, fluorescent dyes,such as styryl dyes, cyanines, rhodamine probes, and oxonols.

The test compound may be contacted neat or as a solution in a solvent.Preferably, the test compound is contacted as a solution. As used hereinthe term “neat” refers to the unmixed or straight technical materialalong with any impurities contained therein. Examples of solvents whichmay be used in the present invention are saline solutions, for examplepotassium, sodium, or magnesium saline solutions, a tissue culturemedia, buffers, for example acidic, basic, or neutral buffers, water, anacid, a ketone, an alcohol, a sulfoxide, or mixtures thereof.Preferably, the test medium is added as a solution in a solvent and iscomprised of a layer of living cells that grow and attach to the testingvessel. The solvents set forth above may also be used in connection withthe test medium.

The time sufficient to allow the membrane potential indicator tointeract with the test medium is preferably in the range of three toabout five hours, more preferably 3.5 to 4.5 hours, at ambienttemperature.

The level of fluorescence of the test medium, which is inverselyproportional to the amount of the test compound acting on the GABA-gatedchloride channel in said testing medium, can be measured by methodsknown to one skilled in the art, such as fluorometric methods, usingequipment know in the art, such as a FLIPR³⁸⁴® Fluorometric ImagingPlate Reader system (available from Molecular Devices Corp.).

In another embodiment of the present invention, a method of identifyinga compound which decreases the amount of fluorescence generated by atest medium is disclosed. The method comprises performing a trialutilizing the method disclosed above and then comparing the results formthe trial to results produced from either:

-   -   (a) a negative control in which no compound is contacted with        the testing medium;    -   (b) a positive control using a positive control compound as the        test compound, wherein the positive control compound is a        compound that decreases the fluorescence of the testing medium;        or    -   (c) both a positive control and a negative control; wherein the        fluorescence of the testing medium is less than the fluorescence        that appears in the testing medium in the negative control and        the fluorescence of the testing medium is more than or equal to        the fluorescence that appears in the testing medium in the        positive control is indicative of a test compound which can        decrease the fluorescence that appears in a testing medium.

Preferably, the results from the trial are compared to results producedfrom both a positive control and a negative control; wherein thefluorescence of the testing medium is less than the fluorescence thatappears in the testing medium in the negative control and thefluorescence of the testing medium is more than or equal to thefluorescence that appears in the testing medium in the positive controlis indicative of a test compound which can decrease the fluorescencethat appears in a testing medium.

Examples of positive control compounds that can be used in the presentinvention are fipronil, endosulfan, dieldrin and picrotoxin. A preferredand economical positive control compound is fipronil.

The method is particularly useful identifying compounds that exhibitinsecticidal activity. The testing vessels, orders of insects, insects,GABA-gated chloride channels, nucleotide and amino acid sequences, andmembrane potential indicators, including, but not limited to, thepreferred testing vessels, orders of insects, insects, GABA-gatedchloride channels, nucleotide and amino acid sequences, and membranepotential indicators, disclosed above can also be used in thisembodiment.

As set forth above, the time sufficient to allow the membrane potentialindicator to interact with the test medium is preferably in the range ofthree to five hours, more preferably 3.5 to 4.5, hours at ambienttemperature. The methods of measuring level of fluorescence of the testmedium set forth above can also be used in this embodiment.

In another embodiment of the present invention, a method of identifyinga compound with insecticidal activity is disclosed. The methodcomprising:

i) performing a trial comprising the steps of:

-   -   (a) contacting a fluorescent dye with a human embryo kidney cell        or cell line transfected with a tobacco budworm GABA-gated        chloride channel having a nucleotide sequence of SEQ. ID NO: 4        in a microtiter plate;    -   (b) maintaining the fluorescent dye in contact with the human        embryo kidney cell or cell line transfected with a tobacco        budworm GABA-gated chloride channel having an amino acid of SEQ.        ID NO: 4 in the microtiter plate for a time sufficient to allow        the fluorescent dye to interact with the human embryo kidney        cell or cell line transfected with a tobacco budworm GABA-gated        chloride channel having a nucleotide sequence of SEQ. ID NO: 4;    -   (c) adding a test compound to the microtiter plate;    -   (d) adding a fixed amount of GABA to the microtiter plate,        wherein both the GABA and test compound desire to act on the        tobacco budworm GABA-gated chloride channel having a nucleotide        sequence of SEQ. ID NO: 4 transfected into the human embryo        kidney cell or cell line;    -   (e) measuring the level of fluorescence of the human embryo        kidney cell or cell line transfected with a tobacco budworm        GABA-gated chloride channel having a nucleotide sequence of SEQ.        ID NO: 4, wherein the level of fluorescence is inversely        proportional to the amount of the test compound acting on the        tobacco budworm GABA-gated chloride channel having a nucleotide        sequence of SEQ. ID NO: 4 transfected into the human embryo        kidney cell or cell line; and

ii) comparing the results from the trial to results produced fromeither:

-   -   (a) a negative control in which no compound is contacted with        the human embryo kidney cell or cell line transfected with a        tobacco budworm GABA-gated chloride channel having a nucleotide        sequence of SEQ. ID NO: 4;    -   (b) a positive control using a positive control compound as the        test compound, wherein the positive control compound is a        compound that decreases the fluorescence of the human embryo        kidney cell or cell line transfected with a tobacco budworm        GABA-gated chloride channel having a nucleotide sequence of SEQ.        ID NO: 4; or    -   (c) both a positive and a negative control; wherein the amount        of fluorescence in the human embryo kidney cell or cell line        transfected with a tobacco budworm GABA-gated chloride channel        having a nucleotide sequence of SEQ. ID NO: 4 is less than the        fluorescence that appears in the human embryo kidney cell or        cell line transfected with a tobacco budworm GABA-gated chloride        channel having a nucleotide sequence of SEQ. ID NO: 4 in the        negative control and the fluorescence of the human embryo kidney        cell or cell line transfected with a tobacco budworm GABA-gated        chloride channel having a nucleotide sequence of SEQ. ID NO: 4        is more than or equal to the fluorescence that appears in the        human embryo kidney cell or cell line transfected with a tobacco        budworm GABA-gated chloride channel having a nucleotide sequence        of SEQ. ID NO: 4 in the positive control is indicative of a test        compound which can decrease the fluorescence that appears in a        testing medium.

The positive control compounds disclosed above can also be used in thisembodiment. Preferably, the positive control compound is fipronil.

Preferably, the time sufficient to allow the dye to interact with thehuman embryo kidney cell or cell line transfected with a tobacco budwormGABA-gated chloride channel having a nucleotide sequence of SEQ. ID NO:4 is in the range of three to four hours at ambient temperature. Similarto the methods disclosed above, the methods of measuring level offluorescence of the test medium set forth above can also be used in thisembodiment.

The level of fluorescence of the human embryo kidney cell or cell linetransfected with a tobacco budworm GABA-gated chloride channel having anucleotide sequence of SEQ. ID NO: 4 may be measured immediately or overa period of time at set intervals. For example, the level offluorescence of the human embryo kidney cell or cell line transfectedwith a tobacco budworm GABA-gated chloride channel having a nucleotidesequence of SEQ. ID NO: 4 may be measured continuously at intervals ofone to ten seconds over a specific period of time. Similar to themethods disclosed above, the methods of and equipment used for measuringthe level of fluorescence of the test medium set forth above can also beused in this embodiment.

The present invention provides an improvement over other methodsdisclosed in the art in that it is a high-throughput method formeasuring a changes in membrane potential of human embryo kidney cell orcell line transfected with a GABA-gated chloride channel derived from aninsect as well as a means of identifying compounds with insecticidalactivity which is less complex, more cost effective, yet comparable insensitivity to those disclosed in the art.

The present invention is now described in more detail by reference tothe following examples, but it should be understood that the inventionis not construed as to be limited thereto.

EXAMPLE 1

This example illustrates the construction of the mammalian expressionvector for a tobacco budworm (“TBW”) GABA-A receptor.

The gene for TBW GABA-A receptor (TBW-a3, SEQ. ID. NO. 4, as disclosedin U.S. Pat. No. 6,329,516 B1, which is incorporated herein byreference) was in a plasmid vector pMT/V5-His A (“pmtALA1”, availablefrom Invitrogen Corporation, Carlsbad, Calif.). In a test tube, pmtALA1was transformed into methylation deficient (dam) bacterial strain, DM1competent cells (available from Life Technologies Inc., Rockville, Md.)by methods know to one of ordinary skill in the art. The bacteria weregrown at 37° C. for about sixteen hours. After this time, the pmtALA1plasmid DNA containing TBW-a3 was isolated using a Qiagen Plasmid MiniKit (available from Qiagen Inc., Valencia, Calif.), yielding isolatedpmtALA1 plasmid DNA containing TBW-a3.

In a separate test tube, 17 μl (0.1 g/μl) of the above pmtALA1 plasmidwas mixed with 2 μl of React 2 buffer (available from Life TechnologiesInc.) and 1 μl of restriction enzyme Xba I (available from LifeTechnologies Inc.). The reaction mixture was incubated at 37° C. for onehour. At the conclusion of this period, the reaction mixture wasseparated on 1% agarose gel and the bands of linearized TBW-a3 DNA wereexcised. The excised TBW-a3 DNA was then purified using a QIAquick GelExtraction Kit (available from Qiagen Inc.), yielding TBW-a3 DNA withXba I sites on both ends.

In another separate test tube, 2 μl (1 ug/μl) of pcDNA3.1 (+) (DNAcontaining a human cytomegalovirus immediate early (CMV) promoter,available from Invitrogen Corporation) was mixed with 2 μl of React 2buffer, 1 μl of Xba I, and 15 μl of water. The reaction mixture wasincubated at 37° C. for 1 hour. At the conclusion of this period, thereaction mixture was separated on 1% agarose gel and the bands oflinearized pcDNA3.1 (+) were excised. The linearized pcDNA3.1 (+) waspurified using a QIAquick Gel Extraction Kit. Upon completion ofpurification, the linearized pcDNA3.1 (+) was dephosphorylated by mixing1 μl (0.7 μg/μl) of the linearized pcDNA3.1 (+) with 4 μl of 10× shrimpalkaline phosphatase (“SAP”) buffer (available from Life TechnologiesInc.), 15 μl of water and 20 μl of SAP (available from Life TechnologiesInc.). The resulting mixture was incubated at 37° C. for 25 minutes, andthen heat-inactivated by incubation at 65° C. for 20 minutes, yieldingdephosphorylated plasmid pcDNA3.1 (+).

In yet another test tube, 1 μl of the above dephosphorylated plasmidpcDNA3.1 (+) was reacted with 6 μl (25 ng/μl) of the TBW-a3 DNA with XbaI sites on both ends, 2 μl of 5× ligase buffer (available fromGIBCO-BRL, Hampstead, N.Y.), and 1 μl of T4 DNA ligase (available fromGIBCO-BRL). The resulting mixture was incubated at 16° C. for aboutsixteen hours. At the conclusion of this period, 1 μl of the resultingmixture was diluted 10 fold in water, and a 1 μl aliquot was transformedinto top 10 chemical competent bacteria by methods know to one ofordinary skill in the art. The transformed bacteria were then replicatedon an agar plate containing LB media. Acceptable clones were selectedand grown by methods know to one of ordinary skill in the art. Theplasmid DNA containing TBW-a3 was isolated using a Qiagen Plasmid MiniKit, yielding the plasmid pcDNA3.1(+) expressing the TBW-a3 GABA-Areceptor, hereinafter referred to as “CMV-GABA-A”. The orientation ofthe CMV-GABA-A was determined by restriction digestion with Apa I.

EXAMPLE 2

This example illustrates the generation of stable HEK cellsconstitutively expressing a TBW GABA-A receptor (hereinafter referred toas “HEK a-3 cells”).

A T75 flask of HEK cells (“HEK293TSA-O cells” available from Cell andMolecular Technologies, Phillipsburg, N.J.) was co-transfected with 2-5μg of the CMV-GABA-A and 2-5 ug of a mammalian expression vectorcontaining a puromycin resistant gene (“pPur Vector”, available from BDBiosciences Clontech, Palo Alto, Calif.) by using a LipofectAMINE PLUS™Reagent package (available from Life Technologies Inc.) according tomanufacturer's instructions. The transfected cells were selected forstable expression by growth in Dulbecco's Modified Eagle Medium(available from ATCC, Manassas, Va.) containing 4 μg/ml of puromycin(available from Clontech Laboratories Inc.) via methods know to one ofordinary skill in the art. A total of 48 resistant clones were grown inDulbecco's Modified Eagle Medium containing 4 μg/ml of puromycin, asdescribed above, and then frozen in two separate vials using liquidnitrogen. In parallel, total RNA was prepared from each resistant cloneusing TRIZOL reagent according to the manufacturer's instruction (LifeTechnologies, Rockville, Md.), and 1 μg of each preparation was spottedonto a Nytran filter (available from Shleicher and Scheull, Keene,N.H.). The filters were hybridized with a radioactive probecorresponding to the open reading frame of GABA-A gene TBW-a3 gene andwashed with an aqueous sodium chloride/sodium citrate/sodium dodecylsulfate solution. For the generation of the radioactive probe,linearized TBW-a3 cDNA was labeled using the T7 random prime labelingkit (Worthington Biochemical Corporation, Lakewood, N.J.) according tothe manufacturer's instruction. The aqueous sodium chloride/sodiumcitrate/sodium dodecyl sulfate solution was prepared by dissolving 2.19grams of sodium chloride, 1.10 grams of sodium citrate and 1 gram ofsodium dodecyl sulfate in 800 ml of water; adjusting pH to 7.0 and thenadjusting the volume to one (1) liter by adding water. After washing,the filter was wrapped in saran wrap, placed against a Kodak film withan intensifying screen on the other side of the film, and placed in anexposure cassette. The exposure cassette was stored at −80° C. for aboutsixteen hours. After this time, the film was developed and 25 cloneswere identified as having detectable levels of the GABA-A mRNAexpression. These 25 clones were then grown in Dulbecco's Modified EagleMedium containing 4 μg/ml of puromycin, as described above, yielding 25clones of HEK a-3 cells that may be used in the assay to measuremembrane potential.

EXAMPLE 3

This example illustrates the measurement of fluorescence in HEK a-3cells in the manner described in the protocol for the FLIPR® MembranePotential Assay Kit.

The following solutions were prepared prior to or on the same day theexperiment was to be carried out:

-   -   Solution A: To about one liter of deionized water was added 8.0        grams of sodium chloride, 0.395 gram of potassium chloride,        0.294 gram of calcium chloride, 0.203 gram of magnesium        chloride, 4.5 grams of glucose, and 2.38 grams of        [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (“HEPES”)        (all available from Aldrich Chemical Company, Milwaukee, Wis.).        The resulting solution was stirred until dissolution was        complete and then the pH adjusted to 7.3 to 7.4 with an aqueous        1 M sodium hydroxide solution.    -   Solution B: To one vile of the dye (“FLIPR® Membrane Potential        Assay Reagent, Component A”) contained in the FLIPR® Membrane        Potential Assay Kit was added about 10 ml of Solution A. The        resulting mixture was mixed by repeated pippetting until the        content of the vile was completely dissolved and used        immediately or stored at 20° C. for future use.

The following solution was prepared on the same day the experiment wasto be carried out:

-   -   Solution C: To solution B was added about 90 mL of Solution A.        The resulting mixture was mixed until dissolution was complete.    -   Solution D: GABA (available from Sigma Chemical Co., St. Louis,        Mo.), 30 grams, was taken up in 30 ml of Solution A. The        resulting solution was mixed until dissolution was completion,        yielding a 10 M GABA solution.    -   Solution E: To 1.2 ml of Solution D was added 38.8 ml of        Solution A. The resulting mixture was mixed by repeated        pippetting until dissolution was complete, yielding a 300 μM        GABA solution.

One day before of the experiment, HEK a-3 cells were seeded into twowells of a poly-D-lysine coated blackwall 96 well microplate with clearbottom at a cell density of about 100,000 cells per well. Uponcompletion of addition, the cells were incubated at 37° C. in a cellculture incubator for twenty-four hours, for use below.

In carrying out the first step, on the day of the experiment, the 96well microplate containing the HEK a-3 cells was removed from theincubator and 100 μl of Solution C was added to the two wells of the 96well microplate. Upon completion of addition, the microplate wasincubated in a cell culture incubator at 37° C. for 0.5 to one hour.After this time, the microplate was placed onto the FLIPR³⁸⁴®Fluorometric Imaging Plate Reader system in which the laser intensitywas set at a suitable level to obtain a basal value of approximately10,000 fluorescence units, fluorescent readings were obtained every onesecond for the first minute and then every six seconds thereafter, andfluorescence measurements were captured by a cooled CCD camera. Afterten seconds from the start of the fluorescence measurements, 25 μl ofSolution E was added at a rate of 25 μl/second by the system to one ofthe two wells of the 96 well microplate. Upon completion of addition,the level of fluorescence of the HEK a-3 cells was determined using theFLIPR³⁸⁴® Fluorometric Imaging Plate Reader system. Please note that nochange in fluorescence was observed when the experiment was run asdescribed above and in the standard protocol for the FLIPR® MembranePotential Assay Kit. See FIG. 1.

EXAMPLE 4

This example illustrates the measurement of fluorescence in HEK a-3cells when the HEK a-3 cells, which interact with a fluorescent dye, areallowed to stand at ambient temperature for an extended period of time.

On the same day the experiment is to be carried, Solutions A-E areprepared again in the manner described above.

One day before of the experiment, HEK a-3 cells were seeded into twowells of a poly-D-lysine coated blackwall 96 well microplate with clearbottom at a cell density of about 100,000 cells per well. Uponcompletion of addition, the cells were incubated at 37° C. in a cellculture incubator for twenty-four hours, for use below.

In carrying out the first step, on the day of the experiment, the 96well microplate containing the HEK a-3 cells was removed from theincubator and 100 μl of Solution C was added to the two wells of the 96well microplate. Upon completion of addition, the microplate was allowedto stand at ambient temperature of 3.5 to 4 hours. After this time, themicroplate was placed onto the FLIPR³⁸⁴® Fluorometric Imaging PlateReader system in which the laser intensity was set at a suitable levelto obtain a basal value of approximately 10,000 fluorescence units,fluorescent readings were obtained every one second for the first minuteand then every six seconds thereafter, and fluorescence measurementswere captured by a cooled CCD camera. After ten seconds from the startof the fluorescence measurements, 25 μl of Solution E was added at arate of 25 μl/second by the system to one of the two wells of the 96well microplate. Upon completion of addition, the level of fluorescenceof the HEK a-3 cells was determined using the FLIPR³⁸⁴® FluorometricImaging Plate Reader system. Please note that an increase influorescence in the HEK a-3 cells was observed when the cells wereallowed to stand at ambient temperature for 3.5 to four hours. See FIG.2.

EXAMPLE 5

This example illustrates the measurement of fluorescence in HEK a-3cells generated from fipronil using a fluorescent dye as the membranepotential indicator.

On the same day the experiment is to be carried, Solutions A-E areprepared again in the manner described above.

One day before of the experiment, HEK a-3 cells were seeded into eachwell of a poly-D-lysine coated blackwall 96 well microplate with clearbottom (available from Becton, Dickinson and Company, Franklin Lakes,N.J.) at a cell density of about 100,000 cells per well. Upon completionof addition, the cells were incubated at 37° C. in a cell cultureincubator for twenty-four hours, for use below.

In carrying out the first step, on the day of the experiment, the 96well microplate containing the HEK a-3 cells was removed from theincubator and 100 μl of Solution C were added to each well of the 96well microplate. Upon completion of addition, the microplate was storedat ambient temperature for 3.5 to 4 hours. After this time, 22 μl ofN,N-dimethylsulfoxide (“DMSO”, available from J. T. Baker Incorporated,final concentration—0.06%) was added to the wells designated as controlwells and 22 μl of various dilutions, ranging in concentration fromabout 0.003 μM to 30 μM, of fipronil (available from Rhone-Poluenc, Inc.Research Triangle Park, N.C.) in DMSO were added to the wells designatedfor testing. Upon completion of addition, the microplate was allowed tostand at ambient temperature for 20 minutes to one hour. After thistime, the microplates were placed onto the FLIPR³⁸⁴® FluorometricImaging Plate Reader system (available from Molecular Devices Corp.) inwhich the laser intensity was set at a suitable level to obtain a basalvalue of approximately 10,000 fluorescence units, fluorescent readingswere obtained every one second for the first minute and then every sixseconds thereafter, and fluorescence measurements were captured by acooled CCD camera. After ten seconds from the start of the fluorescencemeasurements, 25 μl of Solution E were added by the system to the wellsdesignated for testing. Upon completion of addition, the level offluorescence of the HEK a-3 cells was determined using the FLIPR³⁸⁴®Fluorometric Imaging Plate Reader system. See Table 1 for results.

EXAMPLE 6

This example illustrates the measurement of the membrane potential inHEK a-3 cells generated from endosulfan using a fluorescent dye as themembrane potential indicator.

This method was performed in the manner disclosed in Example 5 exceptthat endosulfan was used rather than flipronil. See Table 1 for results.

As the results of Table 1 indicate, the present invention determinedthat all of the test compounds which are known to act on the GABAreceptor decreased the level of fluorescence in the cell line even atlow levels of concentration (see, for example, fipronil, which had 43%inhibition at the 0.01 micromolar (μM) concentration level) where as thecontrols and those compounds which are known not to act on the GABAreceptor did not decrease the fluorescence at all (see, for example,bicuculine, which had 5% inhibition at the 10 micromolar (μM)concentration level). Thus, it will be seen that the present inventioncan be useful in predicting whether or not a prospective compound islikely to exhibit pesticidal activity because as set forth above thegreater the decrease in fluorescence in the cell or cell line the morepesticidal activity a compound may possess.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations in the preferred devices and methods may be used andthat it is intended that the invention may be practiced otherwise thanas specifically described herein. Accordingly, this invention includesall modifications encompassed within the spirit and scope of theinvention as defined by the claims that follow.

The present invention describes an improved method for measuring changesin membrane potential occurring in a human embryo kidney cell or cellline transfected with a GABA-gated chloride channel derived from aninsect, preferably of the order lepidoptera, as well as an improvedmethod for determining insecticidal activity. Said method is animprovement over those disclosed in the art in that it is ahigh-throughput method that is, more cost effective, has betterreproducibility and is comparable in sensitivity to those disclosed inthe art. Said method is also an improvement over the those disclosed inthe art in that it utilizes membrane potential as measured byfluorescence rather than electrophysiological methods or binding abilitythrough the use of a radioisotope or a ligand radiolabeled with adetectable isotope to indicate cellular response. TABLE 1 Fluorescenceand % inhibition of Pesticidally Active Compounds Concentration Compound(μM) Fluorescence⁴ % Inhibition⁵ Control¹ — 12509 — Fipronil² 10 628 953 1031 92 1 1790 86 0.3 2990 76 0.1 4469 64 0.03 6441 48 0.01 8328 430.003 11275 10 Control¹ — 13833 — Endosulfan² 10 993 93 3 1615 88 1 190386 0.3 4426 68 0.1 7098 48 0.03 11074 20 0.01 15198 −9 0.003 14343 −3Control¹ — 13029 — Dieldrin² 10 67 100 3 900 93 1 4184 68 0.3 7279 440.1 9924 34 0.03 11492 12 0.01 12339 5 0.003 13284 −1 Control¹ 12140 —Bicuculine³ 10 11543 5 3 ND — 1 ND — 0.3 ND — 0.1 ND — 0.03 ND — 0.01 ND— 0.003 ND — Control¹ 12140 Pentabarbitol³ 10 11575 5 3 ND — 1 ND — 0.3ND — 0.1 ND — 0.03 ND — 0.01 ND — 0.003 ND —Notes:¹DMSO²Insecticides known to act at the GABA chloride channel³Compounds known not to effect the insect GABA chloride channel⁴Average of three (3) tests⁵% Inhibition refers to the ability of the prospective compound toinhibit the GABA from acting on the available GABA receptors and iscalculated as follows:% Inhibition = [1 − (fluorescence of test compound/fluorescence ofcontrol)] × 100ND indicates no significant difference from control

1. A cell or cell line comprising a human embryo kidney cell or cellline transfected with a GABA-gated chloride channel derived from aninsect.
 2. The cell or cell line of claim 1, wherein the insect is of aorder of Lepidoptera, Diptera, Coleoptera, Homoptera, Acarina,Thysanaptera, Heteroptera, Hymenoptera or Isoptera.
 3. The cell or cellline of claim 1, wherein the GABA-gated chloride channel has anucleotide sequence selected from the group consisting of: SEQ. ID NO:1, SEQ. ID NO: 4, and SEQ. ID NO:
 7. 4. The cell or cell line of claim1, wherein the GABA-gated chloride channel has an amino acid sequenceselected from the group consisting of: SEQ. ID NO: 2, SEQ. ID NO: 5, andSEQ. ID NO.
 8. 5. A cell or cell line comprising a human embryo kidneycell or cell line transfected with a tobacco budworm GABA-gated chloridechannel, wherein the tobacco budworm GABA-gated chloride channel has anucleotide sequence of SEQ. ID NO: 4 or an amino acid sequence of SEQ.ID NO:
 5. 6. A method for measuring levels of membrane potential in atest medium following chemical treatment, wherein the test mediumcomprises a cell or cell line comprising a human embryo kidney cell orcell line transfected with a GABA-gated chloride channel derived from aninsect, the method comprising: (a) contacting a fixed amount of amembrane potential indicator with the test medium in a testing vessel;(b) maintaining the membrane potential indicator in contact with thetest medium in the testing vessel for a time sufficient to allow themembrane potential indicator to interact with the test medium; (c)adding a test compound to the testing vessel; (d) adding a fixed amountof GABA, wherein both the GABA and test compound desire to act on theGABA-gated chloride channel in the test medium; and (e) measuring thelevel of fluorescence of the test medium, wherein the level offluorescence is inversely proportional to the amount of the testcompound acting on the GABA-gated chloride channel in said test medium.7. The method of claim 6, wherein the insect is of a order ofLepidoptera, Diptera, Coleoptera, Homoptera, Acarina, Thysanaptera,Heteroptera, Hymenoptera or Isoptera.
 8. The method of claim 6, whereinthe GABA-gated chloride channel has a nucleotide sequence selected fromthe group consisting of: SEQ. ID NO: 1, SEQ. ID NO: 4, and SEQ. ID NO:7.
 9. The method of claim 6, wherein the GABA-gated chloride channel hasan amino acid sequence selected from the group consisting of: SEQ. IDNO: 2, SEQ. ID NO: 5, and SEQ. ID NO.
 8. 10. The method of claim 6,wherein the GABA-gated chloride channel is a tobacco budworm GABA-gatedchloride channel, wherein the tobacco budworm GABA-gated chloridechannel has a nucleotide sequence of SEQ. ID NO: 4 or an amino acidsequence of SEQ. ID NO:
 5. 11. The method of claim 6, wherein themembrane potential indicator is a fluorescent dye and the time to allowthe membrane potential indicator to interact with the test medium is inthe range of three to five hours at ambient temperature.
 12. A method ofidentifying a compound which decreases the amount of fluorescencegenerated by a test medium, the method comprising: i) performing a trialcomprising the steps of: (a) contacting a fixed amount of a membranepotential indicator with the test medium in a testing vessel, whereinthe test medium comprises a cell or cell line comprising a human embryokidney cell or cell line transfected with a GABA-gated chloride channelderived from an insect; (b) maintaining the membrane potential indicatorin contact with the test medium in the testing vessel for a timesufficient to allow the membrane potential indicator to interact withthe test medium; (c) adding a test compound to the testing vessel; (d)adding a fixed amount of GABA, wherein both the GABA and test compounddesire to act on the GABA-gated chloride channel in the test medium; (e)measuring the level of fluorescence of the test medium, wherein thelevel of fluorescence is inversely proportional to the amount of thetest compound acting on the GABA-gated chloride channel in said testmedium; and ii) comparing the results from the trial to results producedfrom either: (a) a negative control in which no compound is contactedwith the test medium; (b) a positive control using a positive controlcompound as the test compound, wherein the positive control compound isa compound that decreases the fluorescence of the testing medium; or (c)both a positive control and a negative control; wherein the fluorescenceof the testing medium is less than the fluorescence that appears in thetesting medium in the negative control and the fluorescence of thetesting medium is more than or equal to the fluorescence that appears inthe testing medium in the positive control is indicative of a testcompound which can decrease the fluorescence that appears in a testingmedium.
 13. The method of claim 12, wherein said method is used toidentify a compound that exhibits insecticidal activity.
 14. The methodof claim 12, wherein the insect is of a order of Lepidoptera, Diptera,Coleoptera, Homoptera, Acarina, Thysanaptera, Heteroptera, Hymenopteraor Isoptera.
 15. The method of claim 12, wherein the GABA-gated chloridechannel has a nucleotide sequence selected from the group consisting of:SEQ. ID NO: 1, SEQ. ID NO. 4 and SEQ ID. NO:
 7. 16. The method of claim12, wherein the GABA-gated chloride channel has an amino acid sequenceof selected from the group consisting of: SEQ. ID NO: 2, SEQ. ID NO: 5,and SEQ. ID NO:
 8. 17. The method of claim 12, wherein the GABA-gatedchloride channel is a tobacco budworm GABA-gated chloride channel,wherein the tobacco budworm GABA-gated chloride channel has a nucleotidesequence of SEQ. ID NO: 4 or an amino acid sequence of SEQ. ID NO: 5.18. The method of claim 12, wherein the membrane potential indicator isa fluorescent dye and the time to allow the membrane potential indicatorto interact with the test medium is in the range of three to five hoursat ambient temperature.
 19. A method of identifying a compound withinsecticidal activity, the method comprising: i) performing a trialcomprising the steps of: (a) contacting a fluorescent dye with a humanembryo kidney cell or cell line transfected with a tobacco budwormGABA-gated chloride channel having a nucleotide sequence of SEQ. ID NO:4 in a microtiter plate; (b) maintaining the fluorescent dye with thehuman embryo kidney cell or cell line transfected with a tobacco budwormGABA-gated chloride channel having a nucleotide sequence of SEQ. ID NO:4 in the microtiter plate for a time sufficient to allow the fluorescentdye to interact with the human embryo kidney cell or cell linetransfected with a tobacco budworm GABA-gated chloride channel having anucleotide sequence of SEQ. ID NO: 4; (c) adding a test compound to themicrotiter plate; (d) adding a fixed amount of GABA to the microtiterplate, wherein both the GABA and test compound desire to act on thetobacco budworm GABA-gated chloride channel having a nucleotide sequenceof SEQ. ID NO: 4 transfected into the human embryo kidney cell or cellline; (e) measuring the level of fluorescence of the human embryo kidneycell or cell line transfected with a tobacco budworm GABA-gated chloridechannel having a nucleotide sequence of SEQ. ID NO: 4, wherein the levelof fluorescence is inversely proportional to the amount of the testcompound acting on the tobacco budworm GABA-gated chloride channelhaving a nucleotide sequence of SEQ. ID NO: 4 transfected into the humanembryo kidney cell or cell line; and ii) comparing the results from thetrial to results produced from either: (a) a negative control in whichno compound is contacted with the human embryo kidney cell or cell linetransfected with a tobacco budworm GABA-gated chloride channel having anucleotide sequence of SEQ. ID NO: 4; (b) a positive control using apositive control compound as the test compound, wherein the positivecontrol compound is a compound that decreases the fluorescence of thehuman embryo kidney cell or cell line transfected with a tobacco budwormGABA-gated chloride channel having a nucleotide sequence of SEQ. ID NO:4; or (c) both a positive and a negative control; wherein thefluorescence of the human embryo kidney cell or cell line transfectedwith a tobacco budworm GABA-gated chloride channel having a nucleotidesequence of SEQ. ID NO: 4 is less than the fluorescence that appears inthe human embryo kidney cell or cell line transfected with a tobaccobudworm GABA-gated chloride channel having a nucleotide sequence of SEQ.ID NO: 4 in the negative control and the fluorescence of the humanembryo kidney cell or cell line transfected with a tobacco budwormGABA-gated chloride channel having a nucleotide sequence of SEQ. ID NO:4 is more than or equal to the fluorescence that appears in the humanembryo kidney cell or cell line transfected with a tobacco budwormGABA-gated chloride channel having a nucleotide sequence of SEQ. ID NO:4 in the positive control is indicative of a test compound which candecrease the fluorescence that appears in a testing medium.
 20. Themethod of claim 19, wherein the time to allow the fluorescent dye tointeract with the human embryo kidney cell or cell line transfected witha tobacco budworm GABA-gated chloride channel having a nucleotidesequence of SEQ. ID NO: 4 is in the range of three to four hours atambient temperature.